Throughout this application various publications are referenced by full citations within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Pharmacological studies, and more recently gene cloning, have established that multiple receptor subtypes exist for most, if not all, neurotransmitters. The existence of multiple receptor subtypes provides one mechanism by which a single neurotransmitter can elicit distinct cellular responses.
The variation in cellular response can be achieved by the association of individual receptor subtypes with different G proteins and different signalling systems. Further flexibility is provided by the ability of distinct receptors for the same ligand to activate or inhibit the same second messenger system.
Individual receptor subtypes reveal characteristic differences in their abilities to bind a number of ligands, but the structural basis for the distinct ligand-binding properties is not known. Physiologists and pharmacologists have attempted to specify particular biological functions or anatomical locations for some receptor subtypes, but this has met with limited success. Similarly, the biochemical mechanisms by which these receptors transduce signals across the cell surface have been difficult to ascertain without having well-defined cell populations which express exclusively one receptor subtype.
Receptors for serotonin (5-hydroxytryptamine) are termed serotonin or 5-HT receptors. The 5-HT.sub.2 receptor belongs to the family of rhodopsin-like signal transducers which are distinguished by their seven-transmembrane configuration and their functional linkage to G-proteins. While all the receptors of the serotonin type are recognized by serotonin, they are pharmacologically distinct and are encoded by separate genes. These receptors, known as subtypes, are generally coupled to different second messenger pathways that are linked through guanine-nucleotide regulatory (G) proteins. Among the serotonin receptors, 5-HT1.sub.A, 5-HT1.sub.B, and 5-HT1.sub.D receptors inhibit adenylate cyclase, and 5-HT1.sub.C and 5-HT.sub.2 receptors activate phospholipase C pathways, stimulating breakdown of polyphosphoinositides.
Radioligand filtration binding techniques have been employed to characterize the serotonin receptor family (Schmidt and Peroutka, FASEB J. 3:2242 (1989)). Using these methods, at least two classes of G-protein coupled serotonin receptors have been described, 5-HT1, and 5-HT.sub.2. These differ in their selectivity for drugs. 5-HT.sub.1 receptors display high (nanomolar) affinity for serotonin and can be labeled with [.sup.3 H] 5-HT. 5-HT.sub.2 receptors display low affinity for serotonin but have high (nanomolar) affinity for antagonists such as Ketanserin, Mesulergine, Metergoline and d-LSD. Genes for the 5-HT.sub.1A receptor (Fargin, et al., Nature 335:358-360 (1988); Kobilka, et al., Nature 329:75-79 (1987)) and the 5-HT.sub.1C receptor (Julius, et al., Science 241:558-564 (1988)) have been isolated.
Applicants have cloned a human 5-HT.sub.2 receptor, clone 6B, which has been transfected into a heterologous expression system, producing a membrane protein with binding properties consistent with its preliminary characterization based on amino acid homology as the 5-HT.sub.2 receptor subtype. The results from binding studies are consistent with the projected subtype based on amino acid sequence homology.
The receptor encoded by clone 6B shares numerous sequence and structural properties with the family of receptor molecules that has been predicted to span the lipid bilayer seven times. This family includes rhodopsin and related opsins (Nathans, J. and Hogness, D. S., Cell 34:807 (1983)), the .alpha. and .beta. adrenergic receptors (Dohlman, H. G., et al., Biochemistry 26:2657 (1987)), the muscarinic cholinergic receptors (Bonner, T. I., et al., Science 237:527 (1987)), the substance K neuropeptide receptor, (Masu, Y., et al., Nature 329:836 (1987)), the yeast mating factor receptors, (Burkholder, A. C. and Hartwell, L. H., Nucl. Acids Res. 13:8463(1985); Hagan, D. C., et al., Proc. Natl. Acad. Sci. USA 83:1418 (1986)); Nakayama, N. et al., EMBO J. 4:2643 (1985)), the serotonin receptor, and the oncogene c-mas, (Young, et al., Cell 45:711 (1986)). Each of these receptors is thought to transduce extracellular signals by interaction with guanine nucleotide-binding (G) proteins (Dohlman, H. G., et al., Biochemistry 26:2657 (1987); Dohlman, H. G., et al., Biochemistry 27:1813 (1988); O'Dowd, B. F., et al., Ann. Rev. Neurosci., in press).
Membranes of cells transfected with clone 6B bind both .sup.3 H-Ketanserin and .sup.3 H-DOB, demonstrating that the reported "hallucinogen receptor" must be an affinity state of the 5-HT.sub.2 receptor rather than a distinct receptor subtype. Thus, the argument of Titeler (Lyon, et al. Mol. Pharm. 31:194-199 (1987)) for multiple affinity states of the 5-HT.sub.2 receptor is supported and that of Peroutka (Pierce, P. A., and S. J. Peroutka J. Neurochem. 52:656-658 (1989)) for multiple 5-HT.sub.2 receptor subtypes is not. This observation provides the opportunity to use the transfected human 5-HT.sub.2 receptor as a tool for the development of drugs which induce or which interfere with hallucinogenesis, caused either by disease processes or by drugs of abuse.
Strader, Sigal and Dixon recently published a model for the neurotransmitter binding site of G protein-coupled receptors (FASEB J. 3: 1825-1832 (1989)). According to this model, adrenergic receptors contain two serine residues in transmembrane segment V (TM5) which hydrogen bond to the catechol ring hydroxyl groups of adrenergic agonists. Serotonergic receptors, which must bind agonist ligands containing a single ring hydroxyl group, are distinguished by a the presence of a single serine residue in this region of TM5. The rat serotonin 5-HT.sub.2 receptor sequence (Pritchett, et al., EMBO J. 17: 4135-4140 (1988)) shows a single serine residue in this region of TM5, as expected. Surprisingly, the human 5-HT.sub.2 receptor sequence shown in FIG. 2 violates this model by exhibiting two serine residues in this region, as would be expected for an adrenergic receptor. This raises the interesting possibility that the human 5-HT.sub.2 receptor may have evolved the possibility of interacting with epinephrine, norepinephrine and adrenergic antagonists, in addition to its known interactions with serotonergic drugs. This possible acquisition by the human 5-HT.sub.2 receptor of a neurotransmitter cross-reactivity may have functional consequences in the normal or diseased human brain. We hypothesize that the human 5-HT.sub.2 receptor may have evolved the capacity to interact with two separate neurotransmitter systems, the serotonergic and adrenergic systems. Since both systems are widely distributed in the brain and both may act in a neuromodulatory fashion to activate receptors far from the neurotransmitter release site, it is conceivable that the human 5-HT.sub.2 receptor may be activated by a wide array of both serotonergic and adrenergic nerve terminals. In that case, classical adrenergic and serotonergic brain or peripheral nervous system functions may be mediated in part by this single receptor site. Thus, it may be possible to modulate serotonergic functions by administration of adrenergic drugs and to modulate adrenergic functions by administration of serotonergic drugs. These possibilities are currently under investigation in a variety of adrenergic and serotonergic binding, second messenger and physiological response assays.
Another interesting feature of the human 5-HT.sub.2 receptor is the presence of a leucine zipper motif in transmembrane segment I (FIGS. 3A-3B). This motif, consisting of four or more leucine residues repeated every seventh amino acid residue of an alpha-helix, has been implicated as the site of protein--protein interactions in dimerizing, or multisubunit proteins (McCormack et al. Nature 340: 103 (1989)). The presence of this motif in the human 5-HT.sub.2 receptor suggests that this receptor may dimerize in the membrane or may interact with other unidentified proteins (or with G proteins) via the leucine zipper of transmembrane segment I. This may have significant implications for the function of the human 5-HT.sub.2 receptor. In addition, it may be possible to design drugs which interfere with the leucine zipper region of the 5-HT.sub.2 receptor, thus modulating the functional activity of this serotonergic response system.